Exploring Non‐obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions

A 42‐residue polypeptide conjugated to a small‐molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a K_D value of 280 nm . The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l ‐2‐aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the K_D value from 280 to 27 nm . “Downsizing” the 42‐mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a K_D value of 550 nm , comparable with that of the parent 42‐mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the “downsizing” of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.     

Anthracene-Walled Acyclic CB[n] Receptors: in vitro and in vivo Binding Properties toward Drugs of Abuse

We report studies of the interaction of six acyclic CB[n]-type receptors toward a panel of drugs of abuse by a combination of isothermal titration calorimetry and ¹H NMR spectroscopy. Anthracene walled acyclic CB[n] host ( M3 ) displays highest binding affinity toward methamphetamine (K_d=15 nM) and fentanyl (K_d=4 nM). Host M3 is well tolerated by Hep G2 and HEK 293 cells up to 100 μM according to MTS metabolic and adenylate kinase release assays. An in vivo maximum tolerated dose study with Swiss Webster mice showed no adverse effects at the highest dose studied (44.7 mg kg⁻¹). Host M3 is not mutagenic based on the Ames fluctuation test and does not inhibit the hERG ion channel. In vivo efficacy studies showed that pretreatment of mice with M3 significantly reduces the hyperlocomotion after treatment with methamphetamine, but M3 does not function similarly when administered 30 seconds after methamphetamine.     

Small-Molecule Inhibitors Targeting Sterol 14α-Demethylase (CYP51): Synthesis,Molecular Modelling and Evaluation Against Candida albicans

Fungal infections are a global issue affecting over 150 million people worldwide annually, with 750 000 of these caused by invasive Candida infections. Azole drugs are the frontline treatment against fungal infections; however, resistance to current azole antifungals in C. albicans poses a threat to public health. Two series of novel azole derivatives, short and extended derivatives, have been designed, synthesised and investigated for CYP51 inhibitory activity, binding affinity and minimum inhibitory concentration (MIC) against C. albicans strains. The short derivatives were more potent against the C. albicans strains (e. g., MIC 2-(4-chlorophenyl)-N-(2,4-dichlorobenzyl)-3-(1H-imidazol-1-yl)propanamide ( 5 f ) <0.03 μg/mL, N-(4-((4-chlorophenyl)sulfonamido)benzyl)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)propanamide ( 12 c ), 1 μg/mL, fluconazole 0.125 μg/mL) but both displayed comparable enzyme binding and inhibition ( 5 f K_d 62±17 nM, IC₅₀ 0.46 μM; 12 c K_d 43±18 nM, IC₅₀ 0.33 μM, fluconazole K_d 41±13 nM, IC₅₀ 0.31 μM, posaconazole K_d 43±11 nM, IC₅₀ 0.2 μM). The short series had poor selectivity for CaCYP51 over the human homologue, whereas the selectivity of the extended series, for example, compound 12 c , was higher (21.5-fold) than posaconazole (4.7-fold) based on K_d values, although posaconazole was more selective (615-fold) than 12 c (461-fold) based on IC₅₀ values. Based on inhibitory activity and selectivity profile, the extended series are the better of the two series for further development.     

Isolation of a Small‐Molecule Inhibitor of the Antiapoptotic Protein Bcl‐xL from a DNA‐Encoded Chemical Library

Bcl‐xL is an antiapoptotic member of the Bcl‐2 protein family and an attractive target for the development of anticancer agents. Here we describe the isolation of binders to Bcl‐xL from a DNA‐encoded chemical library using affinity‐capture selections and massively parallel high‐throughput sequencing of >30 000 sequence tags of library members. The most potent binder identified, compound 19 / 93 [(R)‐3‐(amido indomethacin)‐4‐(naphthalen‐1‐yl)butanoic acid], bound to Bcl‐xL with a dissociation constant (K_d) of 930 nM and was able to compete with a Bak‐derived BH3 peptide, an antagonist of Bcl‐xL function.     

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K_D=39×10^?9 m ), which is nearly identical to that of the full‐length enzyme (K_D=16×10^?9 m ). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.     

Potent,Metabolically Stable 2‐Alkyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐adenines as Adenosine A2A Receptor Ligands

Inhibition of adenosine A_2A receptors has been shown to elicit a therapeutic response in preclinical animal models of Parkinson’s disease (PD). We previously identified the triazolo‐9H‐purine, ST1535, as a potent A_2AR antagonist. Studies revealed that ST1535 is extensively hydroxylated at the ω‐1 position of the butyl side chain. Here, we describe the synthesis and evaluation of derivatives in which the ω‐1 position has been substituted (F, Me, OH) in order to block metabolism. The stability of the compounds was evaluated in human liver microsomes (HLM), and the affinity for A_2AR was determined. Two compounds, (2‐(3,3‐dimethylbutyl)‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐6‐amine ( 3 b ) and 4‐(6‐amino‐9‐methyl‐8‐(2H‐1,2,3‐triazol‐2‐yl)‐9H‐purin‐2‐yl)‐2‐methylbutan‐2‐ol ( 3 c ), exhibited good affinity against A_2AR (K_i=0.4 nM and 2 nM , respectively) and high in vitro metabolic stability (89.5 % and 95.3 % recovery, respectively, after incubation with HLM for two hours).     

Evaluation of (4‐Arylpiperidin‐1‐yl)cyclopentanecarboxamides As High‐Affinity and Long‐Residence‐Time Antagonists for the CCR2 Receptor

Animal models suggest that the chemokine ligand 2/CC‐chemokine receptor 2 (CCL2/CCR2) axis plays an important role in the development of inflammatory diseases. However, CCR2 antagonists have failed in clinical trials because of a lack of efficacy. We previously described a new approach for the design of CCR2 antagonists by the use of structure–kinetics relationships (SKRs). Herein we report new findings on the structure–affinity relationships (SARs) and SKRs of the reference compound MK‐0483, its diastereomers, and its structural analogues as CCR2 antagonists. The SARs of the 4‐arylpiperidine group suggest that lipophilic hydrogen‐bond‐accepting substituents at the 3‐position are favorable. However, the SKRs suggest that a lipophilic group with a certain size is desired [e.g., 3‐Br: K_i=2.8 nM , residence time (t_res)=243 min; 3‐iPr: K_i=3.6 nM , t_res=266 min]. Alternatively, additional substituents and further optimization of the molecule, while keeping a carboxylic acid at the 3‐position, can also prolong t_res; this was most prominently observed in MK‐0483 (K_i=1.2 nM , t_res=724 min) and a close analogue (K_i=7.8 nM ) with a short residence time.     

Design and Synthesis of Galactosylated Bifurcated Ligands with Nanomolar Affinity for Lectin LecA from Pseudomonas aeruginosa

Lectin A (LecA) from Pseudomonas aeruginosa is an established virulence factor. Glycoclusters that target LecA and are able to compete with human glycoconjugates present on epithelial cells are promising candidates to treat P. aeruginosa infection. A family of 32 glycodendrimers of generation 0 and 1 based on a bifurcated bis‐galactoside motif have been designed to interact with LecA. The influences both of the central multivalent core and of the aglycon of these glycodendrimers on their affinity toward LecA have been evaluated by use of a microarray technique, both qualitatively for rapid screening of the binding properties and also quantitatively (K_d). This has led to high‐affinity LecA ligands with K_d values in the low nanomolar range (K_d=22 nm for the best one).     

Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

Herein we report the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective agonists of the dopamine‐3 (D₃) receptor. A number of these new compounds bind to the D₃ receptor with sub‐nanomolar affinity and show excellent selectivity (>10 000) for the D₃ receptor over the D₁ and D₂ receptors. For example, compound 23 (N‐(cis‐3‐(2‐(((S)‐2‐amino‐4,5,6,7‐tetrahydrobenzo[d]thiazol‐6‐yl)(propyl)amino)ethyl)‐3‐hydroxycyclobutyl)‐3‐(5‐methyl‐1,2,4‐oxadiazol‐3‐yl)benzamide) binds to the D₃ receptor with a K_i value of 0.53 nM and shows a selectivity of >20 000 over the D₂ and D₁ receptors in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes. Moreover, in vitro functional assays showed it to be a full agonist for the human D₃ receptor.     

7,8-Dihydro-8-oxoguanosine Lesions Inhibit the Theophylline Aptamer or Change Its Selectivity

Aptamers are attractive constructs due to their high affinity/selectivity towards a target. Here 7,8-dihydro-8-oxoguanosine (8-oxoG) has been used, due in part to its unique H-bonding capabilities (Watson–Crick or Hoogsteen), to expand the “RNA alphabet”. Its impact on the theophylline RNA aptamer was explored by modifying its binding pocket at positions G11, G25, or G26. Structural probing, with RNases A and T₁, showed that modification at G11 leads to a drastic structural change, whereas the G25-/G26-modified analogues exhibited cleavage patterns similar to that of the canonical construct. The recognition properties towards three xanthine derivatives were then explored through thermophoresis. Modifying the aptamer at position G11 led to binding inhibition. Modification at G25, however, changed the selectivity towards theobromine (K_d≈160 μm ), with a poor affinity for theophylline (K_d>1.5 mm ) being observed. Overall, 8-oxoG can have an impact on the structures of aptamers in a position-dependent manner, leading to altered target selectivity.     

[3H]UR‐DE257: Development of a Tritium‐Labeled Squaramide‐Type Selective Histamine H2 Receptor Antagonist

A series of new piperidinomethylphenoxypropylamine‐type histamine H₂ receptor (H₂R) antagonists with different substituted “urea equivalents” was synthesized and characterized in functional in vitro assays. Based on these data as selection criteria, radiosynthesis of N‐[6‐(3,4‐dioxo‐2‐{3‐[3‐(piperidin‐1‐ylmethyl)phenoxy]propylamino}cyclobut‐1‐enylamino)hexyl]‐(2,3‐³H₂)propionic amide ([³H]UR‐DE257) was performed. The radioligand (specific activity: 63 Ci mmol^?1) had high affinity for human, rat, and guinea pig H₂R (hH₂R, Sf9 cells: K_d, saturation binding: 31 nM , kinetic studies: 20 nM ). UR‐DE257 revealed high H₂R selectivity on membranes of Sf9 cells, expressing the respective hH_xR subtype (K_i values: hH₁R: >10 000 nM , hH₂R: 28 nM , hH₃R: 3800 nM , hH₄R: >10 000 nM ). In spite of insurmountable antagonism, probably due to rebinding of [³H]UR‐DE257 to the H₂R (extended residence time), the title compound proved to be a valuable pharmacological tool for the determination of H₂R affinities in competition binding assays.     

Estimation of interaction between polyanions and bovine serum albumin by means of affinity chromatography

The ligand binding of some polyanions to bovine serum albumin immobilized on Sepharose 4B has been studied by column affinity chromatography. Frontal chromatography using a polyanion of low concentration on an affinity adsorbent gave the dissociation constant K_d of the polyanion-immobilized ligand complex. K_d values determined under various concentrations enabled us to discuss in detail the interactions of bovine serum albumin and polyanions.     

A Single Second Shell Amino Acid Determines Affinity and Kinetics of Linagliptin Binding to Type 4 Dipeptidyl Peptidase and Fibroblast Activation Protein

Drugs targeting type 4 dipeptidyl peptidase (DPP-4) are beneficial for glycemic control, whereas fibroblast activation protein alpha (FAP-α) is a potential target for cancer therapies. Unlike other gliptins, linagliptin displays FAP inhibition. We compared biophysical and structural characteristics of linagliptin binding to DPP-4 and FAP to better understand what differentiates linagliptin from other gliptins. Linagliptin exhibited high binding affinity (K_D) and a slow off-rate (k_off) when dissociating from DPP-4 (K_D 6.6 pM; k_off 5.1×10⁻⁵ s⁻¹), and weaker inhibitory potency to FAP (K_D 301 nM; k_off>1 s⁻¹). Co-structures of linagliptin with DPP-4 or FAP were similar except for one second shell amino acid difference: Asp663 (DPP-4) and Ala657 (FAP). pH dependence of enzymatic activities and binding of linagliptin for DPP-4 and FAP are dependent on this single amino acid difference. While linagliptin may not display any anticancer activity at therapeutic doses, our findings may guide future studies for the development of optimized inhibitors.     

Polypeptide conjugate binders that discriminate between two isoforms of human carbonic anhydrase in human blood

Two binder candidates 4‐C37L34‐B and 3‐C15L8‐B from a 16‐membered set of 42‐residue polypeptide conjugates designed to bind human carbonic anhydrase II (HCAII), were shown to bind HCAII with high affinity in a fluorescence‐based screening assay. Two carbonic anhydrase isoforms with 60 % homology exist in human blood with HCAI being present in five‐ to sevenfold excess over HCAII. The ability of the binders to discriminate between HCAI and HCAII was evaluated with regard to what selectivity could be achieved by the conjugation of polypeptides from a 16‐membered set to a small organic molecule that binds both isoforms with similar affinities. The polypeptide conjugate 4‐C37L34‐B bound HCAII with a K_D of 17 nM and HCAI with a K_D of 470 nM , that is, with a 30‐fold difference in affinity. The corresponding dissociation constants for the complexes formed from 3‐C15L8‐B and the two carbonic anhydrases were 60 and 390 nM , respectively. This demonstration of selectivity between two very similar proteins is striking in view of the fact that the molecular weight of each one of the conjugate molecules is little more than 5000, the fold is unordered, and the polypeptide sequences were designed de novo and have no prior relationship to carbonic anhydrases. The results suggest that synthetic polypeptide conjugates can be prepared from organic molecules that are considered to be weak binders with low selectivity, yielding conjugates with properties that make them attractive alternatives to biologically generated binders in biotechnology and biomedicine.     

Design,Synthesis, and Biological Evaluation of 3‐Benzazepin‐1‐ols as NR2B‐Selective NMDA Receptor Antagonists

Cleavage and reconstitution of a bond in the piperidine ring of ifenprodil ( 1 ) leads to 7‐methoxy‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ols, a novel class of NR2B‐selective NMDA receptor antagonists. The secondary amine 7‐methoxy‐2,3,4,5‐tetrahydro‐1H‐3‐benzazepin‐1‐ol ( 12 ), which was synthesized in six steps starting from 2‐phenylethylamine 3 , represents the central building block for the introduction of several N‐linked residues. A distance of four methylene units between the basic nitrogen atom and the phenyl residue in the side chain results in high NR2B affinity. The 4‐phenylbutyl derivative 13 (WMS‐1405, K_i=5.4 nM ) and the conformationally restricted 4‐phenylcyclohexyl derivative 31 (K_i=10 nM ) represent the most potent NR2B ligands of this series. Whereas 13 shows excellent selectivity, the 4‐phenylcyclohexyl derivative 31 also interacts with σ₁ (K_i=33 nM ) and σ₂ receptors (K_i=82 nM ). In the excitotoxicity assay the phenylbutyl derivative 13 inhibits the glutamate‐induced cytotoxicity with an IC₅₀ value of 360 nM , indicating that 13 is an NMDA antagonist.     

Synthesis and Pharmacological Evaluation of SNC80 Analogues with a Bridged Piperazine Ring

To discover novel δ‐opioid receptor ligands derived from SNC80 ( 1 ), a series of 6,8‐diazabicyclo[3.2.2]nonane derivatives bearing two aromatic moieties was prepared, and the affinity toward δ, μ, and κ receptors, as well as σ receptors, was investigated. After removal of the 4‐methoxybenzyl and 2,4‐dimethoxybenzyl protecting groups, the pharmacophoric N,N‐diethylcarbamoylbenzyl residue was attached to the 6,8‐diazabicyclo[3.2.2]nonane framework to yield the designed δ receptor ligands. In a first series of compounds the benzhydryl moiety of SNC80 was dissected, and one phenyl ring was attached to the bicyclic framework. In a second series of δ ligands the complete benzhydryl moiety was introduced into the bicyclic scaffold. The determined δ receptor affinities show that compounds based on an (R)‐glutamate‐derived bicyclic scaffold possess higher δ receptor affinity than their (S)‐glutamate‐derived counterparts. Furthermore, an intact benzhydryl moiety leads to δ receptor ligands that are more potent than compounds with two separated aromatic moieties. Compound 24 , with the same spatial arrangement of substituents around the benzhydryl stereocenter as SNC80, shows the highest δ receptor affinity of this series: K_i=24 nM . Whereas the highly potent δ ligands reveal good selectivity against μ and κ receptors, the σ₁ and/or σ₂ affinities of some compounds are almost in the same range as their δ receptor affinities, such as compound 25 (σ₂: K_i=83 nM ; δ: K_i=75 nM ). In [³⁵S]GTPγS assays the most potent δ ligands 24 and 25 showed almost the same intrinsic activity as the full agonist SNC80, proving the agonistic activity of 24 and 25 . The enantiomeric 4‐benzylidene derivatives 15 and ent‐ 15 showed selective cytotoxicity toward the 5637 (bladder) and A‐427 (small‐cell lung) human tumor cell lines.     

Improving the Binding Affinity of in‐Vitro‐Evolved Cyclic Peptides by Inserting Atoms into the Macrocycle Backbone

Cyclic peptides binding to targets of interest can be generated efficiently with powerful in vitro display techniques, such as phage display or mRNA display. The cyclic peptide libraries screened with these methods are generated by altering in a combinatorial fashion the amino acid sequence of the peptides, the number of amino acids in the macrocycle rings, and the cyclization chemistry. A structural element that cannot easily be varied in the cyclic peptides is the backbone, which is built from amino acids, each of which contributes three atoms to the macrocyclic ring structure. Here, we proposed to improve the affinity of a phage‐selected bicyclic peptide inhibitor of coagulation factor XII (FXII) by screening variants with one or two carbon atoms inserted into different positions of the backbone, and thus tapping into a structural space that was not sampled by phage display. Two mutants showed 4.7‐ and 2.5‐fold improved K_i values. The better one blocked FXII with a K_i of 1.5±0.1 nm and inhibited activation of the intrinsic coagulation pathway (EC_2x 1.7 μm) . The strategy of ring size variation by one or several atoms should be generally applicable for the affinity maturation of in‐vitro‐evolved cyclic peptides.     

Discovery of a competitive apelin receptor (APJ) antagonist

The apelin receptor (APJ) is a class A G‐protein‐coupled receptor (GPCR) and is a putative target for the treatment of cardiovascular and metabolic diseases. Apelin‐13 (NH₂‐QRPRLSHKGPMPF‐COOH) is a vasoactive peptide and one of the most potent endogenous inotropic agents identified to date. We report the design and discovery of a novel APJ antagonist. By using a bivalent ligand approach, we have designed compounds with two ′affinity′ motifs and a short series of linker groups with different conformational and non‐bonded interaction properties. One of these, cyclo(1–6)CRPRLC‐KH‐cyclo(9–14)CRPRLC is a competitive antagonist at APJ. Radioligand binding in CHO cells transfected with human APJ gave a K_i value of 82 nM , competition binding in human left ventricle gave a K_D value of 3.2 μM , and cAMP accumulation assays in CHO‐K1‐APJ cells gave a K_D value of 1.32 μM .     

Synthesis of σ Receptor Ligands with a Spirocyclic System Connected with a Tetrahydroisoquinoline Moiety via Different Linkers

With the aim to develop new σ₂ receptor ligands, spirocyclic piperidines or cyclohexanamines with 2-benzopyran and 2-benzofuran scaffolds were connected to the 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline moiety by variable linkers. In addition to flexible alkyl chains, linkers containing an amide as functional group were synthesized. The 2-benzopyran and 2-benzofuran scaffold of the spirocyclic compounds were synthesized from 2-bromobenzaldehyde. The amide linkers were constructed by acylation of amines with chloroacetyl chloride and subsequent nucleophilic substitution, the alkyl linkers were obtained by LiAlH₄ reduction of the corresponding amides. For the development of σ₂ receptor ligands, the spirocyclic 2-benzopyran scaffold is more favorable than the ring-contracted 2-benzofuran system. Compounds bearing an alkyl chain as linker generally show higher σ affinity than acyl linkers containing an amide as functional group. A higher σ₁ affinity for the cis-configured cyclohexanamines than for the trans-configured derivatives was found. The highest σ₂ affinity was observed for cis-configured spiro[[2]benzopyran-1,1′-cyclohexan]-4′-amine connected to the tetrahydroisoquinoline system by an ethylene spacer (cis- 31 , K_i (σ₂)=200 nM; the highest σ₁ affinity was recorded for the corresponding 2-benzofuran derivative with a CH₂C=O linker (cis- 29 , K_i (σ₁)=129 nM).